Praveen Awasthi

Multiplex gene editing allows for the simultaneous targeting and mutagenesis of multiple loci in a genome. This tool is particularly valuable for plant genetic improvement, as plant genomes often require mutations at multiple loci to confer useful and/or novel traits. However, the regulation of gene editing can vary depending on the number of loci targeted. In this study, we developed triple‐mutant soybean ( Glycine max (L.) Merrill) lines using different crop improvement strategies, including conventional backcross breeding of standing variant alleles and clustered regularly interspaced short palindromic repeats‐based multiplex editing to introduce new alleles. The mutations were targeted to genes encoding seed antinutritional components, as previously described in a triple null soybean carrying knockout alleles for a Kunitz trypsin inhibitor, a soybean agglutinin, and the allergen P34 protein. The products developed from these respective genetic improvement pipelines were tested for differences between the triple‐mutant lines and their parental lines. Analyses included genomics, seed proteomics, trypsin inhibition, seed protein digestibility, and harvestable yield of the different lines. We observed that both multiplex gene editing and conventional breeding approaches produced essentially equivalent products in comparison to their parental lines. We conclude that the multiplex gene editing strategy is not inherently riskier than conventional breeding for developing complex mutant lines of this type.

The WRKY gene family is widely distributed in plants and plays essential roles in plant development and stress response. Despite extensive characterization of WRKY transcription factors in many plants, a genome-wide analysis of the WRKY gene family in Pisum sativum is still lacking. The central method of this study focused on the retrieval and characterization of WRKY gene sequences in the pea genome and further looking up for their modulation in response to sulfur and water stress. This involved a combination of bioinformatics tools and gene expression study to identify, characterize, and understand the properties of the WRKY gene family in pea. In this study, 86 PsWRKY genes were identified in the pea genome, categorized into five phylogenetic groups. They were dispersed across all seven chromosomes, with Chromosome 5 showing the highest enrichment. Synteny analysis revealed orthologs of 69 PsWRKY genes in Arabidopsis and Medicago. Transcriptome analysis identified 36 differentially expressed PsWRKY genes in response to sulfur (S) stress, water (W) stress, and their combination (W + S). Real-time PCR validation confirmed significant upregulation of PsWRKY23, PsWRKY58, PsWRKY64, and PsWRKY83 under water stress, PsWRKY58 and PsWRKY84 under sulfur stress, and PsWRKY03 under combined (W + S) stress. Overall, this study provides important insights on genetic information of the WRKY gene family in Pea and its possible role in water and sulfur stress, which will help further to study these candidate WRKY gene with a future objective of understanding their role in abiotic stress.

The WRKY gene family is widely distributed in plants and plays essential roles in plant development and stress response. Despite extensive characterization of WRKY transcription factors in many plants, a genome-wide analysis of the WRKY gene family in Pisum sativum is still lacking. The central method of this study focused on the retrieval and characterization of WRKY gene sequences in the pea genome and further looking up for their modulation in response to sulphur and water stress. This involved a combination of bioinformatics tools and gene expression study to identify, characterize, and understand the properties of the WRKY gene family in pea. In this study, 86 PsWRKY genes were identified in the pea genome, categorized into five phylogenetic groups. They were dispersed across all seven chromosomes, with Chromosome 5 showing the highest enrichment. Synteny analysis revealed orthologs of 69 PsWRKY genes in Arabidopsis and Medicago. Transcriptome analysis identified 36 differentially expressed PsWRKY genes in response to sulfur (S) stress, water (W) stress, and their combination (W + S). Real time PCR validation confirmed significant upregulation of PsWRKY23, PsWRKY58, PsWRKY64, and PsWRKY83 under water stress, PsWRKY58 and PsWRKY84 under sulfur stress, and PsWRKY03 under combined (W + S) stress. Overall, this study provides important insights on genetic information of the WRKY gene family in Pea and its possible role in water and Sulphur stress, which will help further to study these candidate WRKY gene with a future objective of understanding their role in abiotic stress.

This study aims to understand the regulatory mechanism of the β-carotene homeostasis by establishing transgene-free genome editing in banana. Carotenoid cleavage dioxygenases (CCDs) belong to a miniature gene family having an imperative role in the intricated carotenoid metabolism in plants. Here, the expression pattern of multiple CCDs was correlated with the levels of carotenoid accumulation in two contrasting cultivars, viz., Nendran (high β-carotene) and Rasthali (low β-carotene). The higher expression of the RAS-CCD4 inversely correlated with β-carotene accumulation in fruit-pulp of the Rasthali. The docking analysis followed enzyme assay of purified RAS-CCD4 suggested β-carotene and 10-apo-β-carotenal as its preferred substrates. Bacterial complementation assay affirmed RAS-CCD4 role in β-carotene degradation and then overexpression of the RAS-CCD4 in the Arabidopsis thaliana further validated results in-vivo by the significant reduction in β-carotene. Subsequently, CRISPR/Cas9 mediated editing of CCD4 was demonstrated in the protoplasts and embryogenic cell lines of Rasthali. The carotenoid profiling in stable mutant lines revealed higher fold β-carotene accumulation in non-green tissue (roots) than in green tissue (leaf) compared with the unedited control plants. The differential expression of carotenoid pathway genes was correlated with the metabolites in the edited lines. The study suggests that carotenoid catabolism is regulated by the CCD4 in tissue and cultivar specific manners, and also demonstrated the use of the genome editing tool in developing transgene-free biofortified banana.

Banana is one of the most important fruit crops consumed globally owing to its high nutritional value. Previously, we demonstrated that the ripe pulp of the banana cultivar (cv.) Nendran (AAB) contained a high amount of pro-vitamin A carotenoids. However, the molecular factors involved in the ripening process in Nendran fruit are unexplored. Hence, we commenced a transcriptome study by using the Illumina HiSeq 2500 at two stages i.e. unripe and ripe fruit-pulp of Nendran. Overall, 3474 up and 4727 down-regulated genes were obtained. A large number of identified transcripts were related to genes involved in ripening, cell wall degradation and aroma formation. Gene ontology analysis highlighted differentially expressed genes that play a key role in various pathways. These pathways were mainly linked to cellular, molecular and biological processes. The present transcriptome study also reveals a crucial role of up-regulated carotenoid biosynthesis pathway genes namely, lycopene beta cyclase and geranylgeranyl pyrophosphate synthase at the ripening stage. Genes related to the ripening and other processes like aroma and flavor were highly expressed in the ripe pulp. Expression of numerous transcription factor family genes was also identified. This study lays a path towards understanding the ripening, carotenoid accumulation and other related processes in banana.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.